Ionizing radiation, topoisomerase inhibitors, radiomimetic drugs, and to some extent all free radical-based genotoxins, induce DNA double-strand breaks (DSBs) that have missing or damaged bases, fragmented sugars, and other modifications at or near the termini. These DSBs cannot be simply religated, but rather require complex processing in order to be rejoined, thus enhancing their potential cytotoxic, clastogenic and mutagenic effects. In G1 and G0-phase cells, the primary pathway for repair of such breaks is nonhomologous end joining, which can join virtually any two DNA ends, regardless of terminal sequence and structure. An in vitro model of this pathway has been developed that combines defined site-specifically labeled substrates, nuclear extracts of human cells, and purified proteins. This in vitro system is capable of accurately restoring the original DNA sequence at the site of a model staggered free radical-mediated DSB, despite missing nucleotides and terminally blocked ends in both strands. The primary goal of the proposed studies is to elucidate the biochemical details of this process, particularly (i) the roles of tyrosyI-DNA phosphodiesterase (hTdpl), Artemis, and the Werner's syndrome factor (Wrn) in processing of protruding and/or recessed 3'-phosphoglycolate termini, (ii) the regulation of such processing by other end-joining factors, and (iii) the structural requirements for accurate gap filling on aligned DSB ends, including the tolerance for oxidatively modified bases and the features of DNA polymerase lambda render it competent for this process. As a complement to these studies, DSB repair and cell survival will be examined in normal and hTdpl-deficient lymphoblastoid cells treated with neocarzinostatin and calicheamicin, agents that specifically induce DSBs with protruding 3'-phosphoglycolate termini, to assess its role in repair in vivo. Such studies may aid in the exploitation of the various end-joining factors as targets for the pharmacological manipulation of DSB repair, with the ultimate goal of improving the efficacy of radio/chemotherapy, and minimizing the genotoxic effects of DSBs.